Studies On Construction And Immune Mechanism Of Recombinant Bb-EmⅡ/3-Em14-3-3 Vaccine Of Echinococcus Multilocularis

ObjectiveTo construct the recombinant Bb-Emâ…¡/3-Em14-3-3 vaccine with Echinococcus multilocularisâ…¡/3 and 14-3-3 antigen coding gene; To analyse the expression of Escherichia.coli-Bifidobacterium shuttle plasmid pGEX-Emâ…¡/3-Em14-3-3 induced by IPTG in E.coli BL21(DE3); To observe dynamically the immune responses of BALB/c mice induced by the recombinant Bb vaccine in subcutaneous injection group and intranasal inoculation group respectively; To study the protective immunity and immune mechanism in BALB/c mice after immunization with the recombinant Bb vaccine against subsequent challenge with protoscoleces by different inoculation routines. This could provide a kind of valuable vaccine to control alveolar echinococcosis .MethodsThe Emâ…¡/3 and Em14-3-3 antigen gene were amplified by PCR and the fusion gene Emâ…¡/3-Em14-3-3 was obtained with gene SOEing technique by splicing Emâ…¡/3 gene and Em14-3-3 gene with (Gly₄Ser)₃,then cloned into Escherichia.coli-Bifidobacterium shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-Emâ…¡/3-Em14-3-3.The recombinant plasmid was introduced into BL21(DE3) and Bb by electroporation to construct recombinant Bb-Emâ…¡/3-Em14-3-3 vaccine.The target protein induced by IPTG in BL21(DE3) was analysed by SDS-PAGE and Western blot.To study the types and dynamic change of immune responses in BALB/c mice after immunization with recombinant Bb-Emâ…¡/3-Em14-3-3 vaccine by subcutaneous injection and intranasal inoculation, 70 female BALB/c mice were randomly divided into 2 groups, 35 in each group. Group of subcutaneous injection: 5×10⁶CFU rBb-Emâ…¡/3-Em14-3-3 vaccines were suspended in 100μl PBS and subcutaneously injected once;Group of intranasal inoculation: 5×10⁵CFU rBb-Emâ…¡/3-Em14-3-3 vaccines were suspended in 10μl PBS and intranasally inoculated once.Serum antibodies of IgG, IgG1, IgG2a, IgG2b, IgG3 and IgE were detected by ELISA at the time of 0, 2, 4, 6, 8, 10, 12 and 14w after immunization. ELISA was used to examine the levels of IFN-γ,IL-12,TNF-αand IL-10 produced by splenocytes with or without Em-Ag and ConA(LPS) stimulation at the above-mentioned time. The proliferation of splenocytes were measured by MTT method, the number of CD₄⁺ and CD₈⁺ subsets of splenocytes were detected by FCM at the above-mentioned time.To study protective immunity and immune mechanism in BALB/c mice after immunization with the recombinant Bb vaccine against subsequent challenge with protoscoleces, 78 female BALB/c mice were randomly divided into 6 groups, 13 in each group. Group SC: 5×10⁶CFU rBb-Emâ…¡/3-Em14-3-3 vaccines were suspended in 100μl PBS and subcutaneously injected once; Group IM: 5×10⁶CFU rBb-Emâ…¡/3-Em14-3-3 vaccines were suspended in 100μl PBS and intramuscularly injected once; Group IN: 5×10⁵CFU rBb-Emâ…¡/3-Em14-3-3 vaccines were suspended in 10μl PBS and intranasally inoculated once;Group PO: 1×10⁸CFU rBb-Emâ…¡/3-Em14-3-3 vaccines were suspended in 60μl PBS and inoculated per os once; Group Bb: Bb was subcutaneously injected; Group PBS: 100μl PBS served subcutaneously as control. All the mice were challenged with protoscoleces of Echinococcus multilocularis on 12w after immunization. On the 18th week after infection,Serum antibodies of IgG, IgG1, IgG2a, IgG2b,IgG3 and IgE were respectively detected by ELISA. Sandwich ELISA kits were used to examine the levels of IFN-γ, IL-12, TNF-αand IL-10 in splenocytes with or without Em-Ag and ConA (LPS) stimulation. The proliferation of splenocytes were measured by MTT method, the number of CD₄⁺ and CD₈⁺ subsets of spleen lymphocyte were detected by FCM and the apoptotic rate of splenocytes with or without ConA stimulation were examined by Annexin V-FITC kits.ResultsEmâ…¡/3-Em14-3-3 fusion gene identified by Agarose gel electrophoresis was about 2554bp,which was in accordance with the expected result by sequence analyzing.Restriction-endonuclease digestion demonstrated that Emâ…¡/3-Em14-3-3 fusion gene was correctly cloned into the multiple cloning site of Escherichia.coli-Bifidobacterium shuttle expression plasmid pGEX-1λT to construct recombinant plasmid pGEX-Emâ…¡/3-Em14-3-3. It was confirmed that the recombinant plasmid was successfully introduced into Bb by analyzing the rBb selected in MRS medium with 50μg/ml Amp by PCR and restriction-endonuclease digestion.SDS-PAGE and Western blot showed the recombinant plasmid was able to express target protein in E.coli BL21(DE3) inducing by IPTG for 1¹3h,which was expressed as a band of 119kDa.The content contained 20% of total bacterial protein of E.coli and could combine with specific antibody in mice serum.The dynamic observation showed that in 2 groups, the level of IgG, IgG2a and IgG2b antibodies in sera showed various degree of increase after immunization, that of IgE obviously increased from 10 to 12 week, but that of IgG1 and IgG3 had no obvious change; the proliferation of splenocytes was high level from 10 to 14 week; CD₄⁺T cells were high level from 4 to 8 week and CD₈⁺T cells sightly increased; 4 cytokines secreted by splenocytes with or without EmAg and ConA (LPS) stimulation were high level from 2 to 6 week, both of IFN-γand TNF-αincreased remarkablely.On the 18th week after mice challenged with protoscoleces,in the immunized groups, the rate of reduced alveolar echinococcus weight was 13.56%⁷8.25%, and that in the group of subcutaneous injection was highest; the level of IgG,IgG2a,IgG2b and IgG1 were remarkably higher than PBS control, that of IgG,IgG2a and IgG2b in the group of subcutaneously injected were remarkably higher than the other immunized groups, that of IgG3 and IgE obviously decreased; the proliferation of splenocytes with or without EmAg and ConA stimulation were remarkably higher than PBS control; CD4 + T cells increased apparently but the change of CD₈⁺T had no special meaning between immunized groups and control; the level of IL-12,IFN-γand TNF-αincreased obviously while that of IL-10 decreased remarkably; the apoptotic rate of splenocytes with or without ConA stimulation in the immunized groups were remarkably lower than PBS control,and that in the group of subcutaneous injection was lowest.Conclusion1. The Emâ…¡/3-Em14-3-3 fusion gene was successfully amplified by Gene SOEing technique.2. The shuttle expression plasmid pGEX-Emâ…¡/3-Em14-3-3 and the recombinant vaccine Bb-Emâ…¡/3-Em14-3-3 was successfully constructed. 3. The recombinant plasmid pGEX-Emâ…¡/3-Em14-3-3 was able to express 20 percent target protein in E.coli BL21(DE3) induced by IPTG,which was of specific antigenicity.4. The recombinant Bb-Emâ…¡/3-Em14-3-3 vaccine is able to elicit strong specific CD₄⁺Th1 cellular immune , humoral immune and weak CD₈⁺CTL respones.5. The protective immunity is able to be induced by the rBb-Emâ…¡/3-Em14-3-3 vaccine against challenge with protoscoleces, and the protective immunity induced by subcutaneous injection is better than that of other immunized groups.